The principal objective of this study is to determine the effect of ethanol on the metabolism of neurosteroids in the central nervous system. Neurosteroids such as 5a-pregnane-3a-ol-20-one (THP or Allopregnanolone) and 5a-pregnane-3a,21-diol-20-one (THDOC) have been shown to modulate the GABA/benzodiazepine binding sites and to exert anxiolytic and hypnotic effects. Modulation of these neurosteroids in the central nervous system by ethanol may be one of the underlying mechanisms for stress-related situations in humans often observed in alcoholics during withdrawal. As a continuing effort from the last year's establishment of a sensitive mass spectrometric technique to measure trace levels of neurosteroids, we characterized their metabolism in astroglia where most neurosteroids are synthesized. In C-6 glioma, which retain most of the astrocytic characteristics, progesterone metabolized to 5a-DHP (dihydroprogesterone), 3a and 3b-THP, as well as P-450 metabolites. Using a sensitive technique which has been developed in our laboratory to detect trace levels of neurosteroids using electrospray liquid chromatography/mass spectrometry (LC/MS), their structure and level could be determined. Prolonged exposure of the cells to ethanol for 3 weeks at as low as 10 mM concentration significantly increased the production of 3a-THP and decreased 3b-THP and P-450 metabolites while accumulating the precursor progesterone and 5a-DHP. Rat brain cortex also metabolized progesterone to 5a-DHP and THP. After exposure to ethanol for 24 days, changes in the metabolism by rat brain cortex showed the same trend as was observed with C-6 glioma cells. However, these changes were not statistically significant, probably due to the insufficient duration of the ethanol exposure. Investigation of the changes in the level of neuroactive steroids in rat brain as well as in human CSF after ethanol exposure is now in progress.